With the depletion of petrochemical fuels, intensive worldwide attention has been paid to alternative energies. As one of the alternative energies, fuel ethanol can be converted from a cellulosic biomass. A tremendous quantity of cellulose is produced each year as they are fixed through photosynthesis. In addition, cellulose is regenerable. When account is taken of its regenerability and productivity, cellulose is very advantageous functionally and economically. These days, a lignocellulosic biomass is predominantly utilized out of the cellulosic biomass, and extensive research has been focused on the effective degradation of the ingredients of a lignocellulosic biomass including cellulose, hemicellulose, and lignin; additionally a search for new strains of cellulosic biomass producers, and saccharification and fermentation processes are being researched.
Production of cellulosic fuels may be largely divided into i) an enzymatic saccharification process of a biomass using at least three enzymes (endoglucanase, exoglucanase, and β-glucanase), and ii) a microbial fermentation process of the sugars thus obtained. Recently, there have been extensive studies on simultaneous saccharification and fermentation (SSF) that is designed to simultaneously perform an enzymatic saccharification process and a fermentation process in one reactor whereby a significant reduction can be brought about in facility cost and enzymatic inhibitory activity, resulting in an increase in the production efficiency of ethanol. Of the processes, the enzymatic saccharification process is the most costly. Thus, studies have been directed towards either the functional enhancement or the use reduction of the enzymes used in saccharification by developing fermentation strains of bacteria which produce pertinent enzymes. Particularly, a recent advance in bioengineering technology has allowed genes of saccharification-related enzymes to be introduced into and expressed in fermentation strains of bacteria, in order to develop strains of bacteria capable of simultaneously performing saccharification and fermentation. However, this strategy suffers from the disadvantage of being very low in the expression efficiency of exogenous genes such as saccharification-related genes, and having a negative influence on cell growth and metabolism upon overexpression. Hence, the focus of interest has been shifted from the introduction of exogenous genes to a modification in the regulation of pathways endogenous to fermentation strains.
Escherichia coli is regarded as an efficient means for the production of lignocellulosic fuels because it can utilize all of the sugars present in hydrolysates of a biomass. However, if a preferred sugar (e.g., glucose) exists in the hydrolysates, carbon catabolite repression (CCR), which accounts for the inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the target, occurs with the consequent restriction of the potentiality of microorganisms. Sugars such as xylose and arabinose, although present in hydrolysates, cannot be metabolized until glucose is completely depleted. This preference for glucose disturbs fermentation processes thereby reducing the efficiency of the processes, and has a negative effect on downstream processes due to the accumulation of unutilized carbon sources. Sugar mixtures obtained from lignocellulosic hydrolysates are highly variable in composition, but with a predominance of glucose and xylose over other sugars. To improve the production of cellulosic fuels in terms of cost, efficiency and ease, there is a requirement for the development of a mutant E. coli which can utilize these two sugars simultaneously.
The simultaneous utilization of glucose and xylose has been demonstrated by some catabolite derepressed E. coli strains. Many studies have been focused on cAMP receptor protein (CRP), known as a global transcriptional regulator of CCR. Some E. coli strains with mutant CRP (CRP*) were found to partially deviate from the CCR control (Karimova, G., et al., Research in Microbiology, 2004, 155(2): 76-79; Nair, N. U., et al., Metabolic Engineering, 2010, 12(5): 462-468; Kimata, K., et al., Proceedings of the National Academy of Sciences of the Unites States of America, 1997, 94(24): 12914-12919; Inada, T., et al., Genes Cells, 1996, 1(3): 293-301). In addition, glucose phosphotransferase system (PTS)-devoid of E. coli was partially deprived of CCR, and the deletion of methylglyoxal synthase gene was helpful in regulating the pattern of utility of glucose. However, these methods cannot remove CCR to a sufficient enough extent to produce cellulosic biofuels, and accordingly, there still remains a need for new approaches.
Meanwhile, L-arabinose metabolism-related operons and genes, generally referred to as the ara operons, are gene sequence encoding enzymes needed for the catabolism of arabinose to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. Among the ara operons are the araBAD operon and the araFGH operon, and araE as an individual gene. Of them, araB codes for L-ribulokinase, araA for L-arabinose isomerase, araD for L-ribulose-5-phosphatase 4-epimerase; araF, araG and araH for respective arabinose ABC transporter subunits; and araE for arabinose/hydrogen ion symporter (Mayer, C. and W. Boos, Chapter 3.4.1, Hexose/Pentose and Hexitol/Pentitol Metabolism. In A. Böck, R. Curtiss III, J. B. Kaper, P. D. Karp, F. C. Neidhardt, T. Nyström, J. M. Slauch, C. L. Squires, and D. Ussery (ed.), EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology. http://www.ecosal.org. ASM Press, Washington, D.C.).
Likewise, D-xylose metabolism-related operons and genes, which are generally called xyl operons, are gene sequence encoding enzymes needed for the catabolism of xylose to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The xyl operon contains xylAB operon and xylGFH operon wherein xylA codes for D-xylose isomerase, xylB for xylulokinase, and xylF, xylG and xylH for respective D-xylose ABC transferase subunits (Mayer, C. and W. Boos, Chapter 3.4.1, Hexose/Pentose and Hexitol/Pentitol Metabolism. In A. Böck, R. Curtiss III, J. B. Kaper, P. D. Karp, F. C. Neidhardt, T. Nyström, J. M. Slauch, C. L. Squires, and D. Ussery (ed.), EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology. http://www.ecosal.org. ASM Press, Washington, D.C.)
Leading to the present invention, intensive and thorough research into the effective production of a biofuel, a biologically active ingredient, and a medicinal material from a biomass resulted in the finding that when inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon, and xylGFH operon in a wild-type E. coli were changed into constitutive ones, the resulting mutant E. coli grown in a xylose minimal medium or an arabinose and xylose minimal medium, could utilize glucose and xylose, simultaneously.